Block of Kcnk3 by protons - Evidence that 2-p-domain potassium channel subunits function as homodimers

KCNK subunits have two pore-forming P domains and four predicted transmembr ane segments. To assess the number of subunits in each pore, we studied ext ernal proton block of Kcnk3, a subunit prominent in rodent heart and brain. Consistent with a pore-blocking mechanism, inhibition was dependent on vol tage, potassium concentration, and a histidine in the first P domain (P1H). Thus, at pH 6.8 with 20 mM potassium half the current passed by P1H channe ls was blocked (apparently via two sites similar to 10% into the electrical field) whereas channels with an asparagine substitution (P1N) were fully a ctive. Furthermore, pore blockade by barium was sensitive to pH in P1H but not P1N channels. Although linking two Kcnk3 subunits in tandem to produce P1H-P1H and P1N-P1N channels bearing four P domains did not alter these att ributes, the mixed tandems P1H-P1N and P1N-P1H were half-blocked at pH simi lar to6.4, apparently via a single site. This implicates a dimeric structur e for Kcnk3 channels with two (and only two) P1 domains in each pore and ar gues that P2 domains also contribute to pore formation.